The invention relates to novel alkenyl and alkynyl compounds, alone or as part of a composition, which are potent and highly selective inhibitors of factor Xa isolated or assembled in the prothrombinase complex. Such compounds show selectivity for factor Xa over other proteases of the coagulation (e.g., thrombin, fvhla, fixa) or the fibrinolytic cascades (e.g., plasminogen activators, plasmin). The invention further relates to methods for using such alkenyl and alkynyl compounds as agents for preventing or treating a condition in a mammal characterized by undesired coagulation disorders, such as thrombosis.
Hemostasis, the control of bleeding, occurs by surgical means, or by the physiological properties of vasoconstriction and coagulation. Under normal hemostatic circumstances, the body maintains an acute balance of clot formation and clot removal (fibrinolysis). The blood coagulation cascade involves the conversion of a variety of inactive enzymes (zymogens) into active enzymes which ultimately convert the soluble plasma protein fibrinogen into an insoluble matrix of highly cross-linked fibrin. Davie, E.J. et al., xe2x80x9dThe Coagulation Cascade: Initiation, Maintenance and Regulationxe2x80x9d, Biochemistry, 30, 10363-10370 (1991). These plasma glycoprotein zymogens include Factor XII, Factor XI, Factor IX, Factor X, Factor VII, and prothrombin. Blood coagulation follows either the intrinsic pathway, where all of the protein components are present in blood, or the extrinsic pathway, where the cell-membrane protein tissue factor plays a critical role. Clot formation occurs when fibrinogen is cleaved by thrombin to form fibrin. Blood clots are composed of activated platelets and fibrin.
Blood platelets which adhere to damaged blood vessels are activated and incorporated into the clot and thus play a major role in the initial formation and stabilization of hemostatic xe2x80x9cplugsxe2x80x9d. In certain diseases of the cardiovascular system, deviations from normal hemostasis push the balance of clot formation and clot dissolution towards life-threatening thrombus formation when thrombi occlude blood flow in coronary vessels (myocardial infarctions) or limb and pulmonary veins (venous thrombosis). Although platelets and blood coagulation are both involved in thrombus formation, certain components of the coagulation cascade are primarily responsible for the amplification or acceleration of the processes involved in platelet aggregation and fibrin deposition.
Thrombin is a key enzyme in the coagulation cascade as well as in hemostasis. Thrombin plays a central role in thrombosis through its ability to catalyze the conversion of fibrinogen into fibrin and through its potent platelet activation activity. Under normal circumstances, thrombin can also play an anticoagulant role in hemostasis through its ability to convert protein C into activated protein C (aPC) in a thrombomodulin-dependent manner. However, in atherosclerotic arteries these thrombin activities can initiate the formation of a thrombus, which is a major factor in pathogenesis of vasoocclusive conditions such as myocardial infarction, unstable angina, nonhemorrhagic stroke and reocclusion of coronary arteries after angioplasty or thrombolytic therapy. Thrombin is also a potent inducer of smooth muscle cell proliferation and may therefore be involved in a variety of proliferative responses such as restenosis after angioplasty and graft induced atherosclerosis. In addition, thrombin is chemotactic for leukocytes and may therefore play a role in inflammation. (Hoover, R.J., et al. Cell, 4, 423 (1978); Etingin, O. R., et al., Cell, 6 657 (1990). These observations indicate that inhibition of thrombin formation or inhibition of thrombin itself may be effective in preventing or treating thrombosis, limiting restenosis and controlling inflammation.
Direct or indirect inhibition of thrombin activity has been the focus of a variety of recent anticoagulant strategies as reviewed by Claeson, G., xe2x80x9cSynthetic Peptides and Peptidomimetics as Substrates and Inhibitors of Thrombin and Other Proteases in the Blood Coagulation Systemxe2x80x9d, Blood Coag. Fibrinol. 5, 411-436 (1994). Several classes of anticoagulants currently used in the clinic directly or indirectly affect thrombin (i.e. heparins, low-molecular weight heparins, heparin-like compounds and coumarins).
The formation of thrombin is the result of the proteolytic cleavage of its precursor prothrombin at the Arg-Thr linkage at positions 271-272 and the Arg-Ile linkage at positions 320-321. This activation is catalyzed by the prothrombinase complex, which is assembled on the membrane surfaces of platelets, monocytes, and endothelial cells. The complex consists of Factor Xa (a serine protease), Factor Va (a cofactor), calcium ions and the acidic phospholipid surface. Factor Xa is the activated form of its precursor, Factor X, which is secreted by the liver as a 58 kd precursor and is converted to the active form, Factor Xa, in both the extrinsic and intrinsic blood coagulation pathways. Factor X is a member of the calcium ion binding, gamma carboxyglutamyl (Gla)-containing, vitamin K dependent, blood coagulation glycoprotein family, which also includes Factors VII and IX, prothrombin, protein C and protein S (Furie, B., et al., Cell, 53, 505 (1988)). The activity of Factor Xa in effecting the conversion of prothrombin to thrombin is dependent on its inclusion in the prothrombinase complex.
The prothrombinase complex converts the zymogen prothrombin into the active procoagulant thrombin. It is therefore understood that Factor Xa catalyzes the next-to- last step in the blood coagulation cascade, namely the formation of the serine protease thrombin. In turn, thrombin then acts to cleave soluble fibrinogen in the plasma to form insoluble fibrin.
The location of the prothrombinase complex at the convergence of the intrinsic and extrinsic coagulation pathways, and the resulting significant amplification of thrombin generation (several hundred-thousand fold faster in effecting the conversion of prothrombin to thrombin than Factor Xa in soluble form) mediated by the complex at a limited number of targeted catalytic units present at vascular lesion sites, suggests that inhibition of thrombin generation is a desirable method to block uncontrolled procoagulant activity. It has been suggested that compounds which selectively inhibit factor Xa may be useful as in vitro diagnostic agents, or for therapeutic administration in certain thrombotic disorders, see e.g., WO 94/13693. Unlike thrombin, which acts on a variety of protein substrates as well as at a specific receptor, factor Xa appears to have a single physiologic substrate, namely prothrombin.
Plasma contains an endogenous inhibitor of both the factor Vlla-tissue factor (TF) complex and factor Xa called tissue factor pathway inhibitor (TFPI). TFPI is a Kunitz-type protease inhibitor with three tandem Kunitz domains. TFPI inhibits the TF/fVIIa complex in a two-step mechanism which includes the initial interaction of the second Kunitz domain of TFPI with the active site of factor Xa, thereby inhibiting the proteolytic activity of factor Xa. The second step involves the inhibition of the TF/fVIIa complex by formation of a quaternary complex TF/fVIIa/TFPI/fXa as described by Girard, T. J. et aL, xe2x80x9cFunctional Significance of the Kunitz-type Inhibitory Domains of Lipoprotein-associated Coagulation Inhibitorxe2x80x9d, Nature, 3, 518-520 (1989).
Polypeptides derived from hematophagous organisms have been reported which are highly potent and specific inhibitors of factor Xa, e.g., U.S. Pat. No. 4,588,587. Also, Factor Xa inhibitory compounds which are not large polypeptide-type inhibitors have also been reported, e.g., Tidwell, R. R. et al., xe2x80x9cStrategies for Anticoagulation With Synthetic Protease Inhibitors. Xa Inhibitors Versus Thrombin Inhibitorsxe2x80x9d, Thromb. Res., 19, 339-349 (1980)
Accordingly, there exists a need in the art for effective therapeutic agents for the regulation of hemostasis. There also exists a need in the art for effective therapeutic agents for the prevention and treatment of thrombus formation and other pathological processes in the vasculature induced by thrombin such as, for example, restenosis and inflammation. Particularly needed are small molecule antagonists or inhibitors of Factor X or of its activated form Factor Xa. The invention as described below answers this need.
The invention relates to alkenyl and alkynyl compounds capable of assisting in maintaining the integrity of mammalian circulation after injury, inflammation, disease, congenital defect, dysfunction or other disruption. In particular, the invention provides alkenyl and alkynyl compounds, including their pharmaceutically acceptable isomers, salts, hydrates, solvates and prodrug derivatives, having certain biological properties and useful as potent and specific inhibitors of blood coagulation. The alkenyl and alkynyl compounds of the invention are potent and highly selective inhibitors of isolated factor Xa when assembled in the prothrombinase complex. The compounds of the invention exhibit selectivity for factor Xa over other proteases of the coagulation cascade (e.g., thrombin) or the fibrinolytic cascade.
The invention also provides pharmaceutical compositions comprising an alkenyl or alkynyl compound of the invention and a pharmaceutically acceptable carrier.
The compounds of the invention are useful as diagnostic reagents as well as antithrombotic agents. Accordingly, the invention further provides a method of using the compounds of the invention in the diagnosis, treatment and/or prevention of a condition in a mammal characterized by undesired coagulation disorders (e.g., thrombotically mediated acute coronary or cerebrovascular syndrome, thrombotic syndrome occurring in the venous system, coagulopathy, and any thrombotic complications associated with extracorporeal circulation or instrumentation).
The invention still further relates to a method of using the compounds of the invention for the inhibition of coagulation in biological samples.
In accordance with the invention and as used herein, the following terms are defmed with the following meanings, unless explicitly stated otherwise.
The term xe2x80x9calkenylxe2x80x9d as used herein refers to a trivalent straight chain or branched chain unsaturated aliphatic radical radical that includes at least two carbons joined by a double bond. The term xe2x80x9calkinylxe2x80x9d (or xe2x80x9calkynylxe2x80x9d) as used herein refers to a straight or branched chain aliphatic radical that includes at least two carbons joined by a triple bond. If no number of carbons is specified, xe2x80x9calkenylxe2x80x9d and xe2x80x9calkinylxe2x80x9d each refer to radicals having from 2-12 carbon atoms.
The term xe2x80x9calkylxe2x80x9d as used herein refers to saturated aliphatic groups including straight-chain (i.e. linear) and branched-chain groups having the number of carbon atoms specified, or if no number is specified, having up to 12 carbon atoms. The term xe2x80x9ccycloalkylxe2x80x9d as used herein refers to a mono-, bi-, or tricyclic aliphatic ring system having 3 to 14 carbon atoms, preferably, 3 to 7 carbon atoms.
As used herein, the terms xe2x80x9ccarbocyclic ring structurexe2x80x9d and xe2x80x9cC3-6 carbocyclic mono, bicyclic or tricyclic ring structurexe2x80x9d or the like are each intended to mean stable ring structures having only carbon atoms as ring atoms wherein the ring structure is a substituted or unsubstituted member selected from the group consisting of a stable monocyclic ring which is aromatic ring (xe2x80x9carylxe2x80x9d) having six ring atoms; a stable monocyclic non-aromatic ring having from 3 to 7 ring atoms in the ring; a stable bicyclic ring structure having a total of from 7 to 12 ring atoms in the two rings wherein the bicyclic ring structure is selected from the group consisting of ring structures in which both of the rings are aromatic, ring structures in which one of the rings is aromatic and ring structures in which both of the rings are non-aromatic; and a stable tricyclic ring structure having a total of from 10 to 16 atoms in the three rings wherein the tricyclic ring structure is selected from the group consisting of ring structures in which three of the rings are aromatic, ring structures in which two of the rings are aromatic and ring structures in which three of the rings are non-aromatic. In each case, the non-aromatic rings when present in the monocyclic, bicyclic or tricyclic ring structure may independently be saturated, partially saturated or fully saturated. Examples of such carbocyclic ring structures include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), 2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adarnantyl, or tetrahydronaphthyl (tetralin). Moreover, the ring structures described herein may be attached to one or more indicated pendant groups via any carbon atom which results in a stable structure. The term xe2x80x9csubstitutedxe2x80x9d as used in conjunction with carbocyclic ring structures means that hydrogen atoms attached to the ring carbon atoms of ring structures described herein may be substituted by one or more of the groups indicated for that structure if such substitution(s) would result in a stable compound.
The term xe2x80x9carylalkylxe2x80x9d which is included with the term xe2x80x9ccarbocyclic arylxe2x80x9d as used herein refers to one, two, or three aryl groups having the number of carbon atoms designated, appended to an alkyl group having the number of carbon atoms designated. Suitable arylalkyl groups include, but are not limited to, benzyl, picolyl, naphthylmethyl, phenethyl, benzyhydryl, trityl, and the like, all of which may be optionally substituted.
As used herein, the term xe2x80x9cheterocyclic ringxe2x80x9d or xe2x80x9cheterocyclic ring systemxe2x80x9d is intended to mean a substituted or unsubstituted member selected from the group consisting of stable monocyclic ring having from 5-7 members in the ring itself and having from 1-4 heteroatoms in the ring selected from N, O and S; a stable bicyclic ring structure having a total of from 7 to 12 atoms in the two rings wherein at least one of the two rings has from 1-4 heteroatoms selected from N, O and S, including bicyclic ring structures wherein any of the described stable monocyclic heterocyclic rings is fused to a hexane or benzene ring; and a stable tricyclic heterocyclic ring structure having a total of from 10 to 16 atoms in the three rings wherein at least one of the three rings has from 1-4 heteroatoms selected from N, O and S. Any nitrogen and sulfur atoms present in a heterocyclic ring of such a heterocyclic ring structure may be oxidized. Unless indicated otherwise the terms xe2x80x9cheterocyclic ringxe2x80x9d or xe2x80x9cheterocyclic ring systemxe2x80x9d include aromatic rings, as well as non-aromatic rings which can be saturated, partially saturated or fully saturated non-aromatic rings. Also, unless indicated otherwise the term xe2x80x9cheterocyclic ring systemxe2x80x9d includes ring structures wherein all of the rings contain at least one hetero atom as well as structures having less than all of the rings in the ring structure containing at least one hetero atom, for example bicyclic ring structures wherein one ring is a benzene ring and one of the rings has one or more hetero atoms are included within the term xe2x80x9cheterocyclic ring systemsxe2x80x9d as well as bicyclic ring structures wherein each of the two rings has at least one hetero atom. Moreover, the ring structures described herein may be attached to one or more indicated pendant groups via any hetero atom or carbon atom which results in a stable structure. Further, the term xe2x80x9csubstitutedxe2x80x9d means that one or more of the hydrogen atoms on the ring carbon atom(s) or nitrogen atom(s) of the each of the rings in the ring structures described herein may be replaced by one or more of the indicated groups if such replacement(s) would result in a stable compound. Nitrogen atoms in a ring structure may be quaternized, but such compounds are specifically indicated or are included within the term xe2x80x9ca pharmaceutically acceptable saltxe2x80x9d for a particular compound. When the total number of O and S atoms in a single heterocyclic ring is greater than 1, it is preferred that such atoms not be adjacent to one another. Preferably, there are no more that 1 O or S ring atoms in the same ring of a given heterocyclic ring structure.
Examples of monocyclic and bicyclic heterocyclic ring systems, in alphabetical order, are acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyroazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pryidooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Preferred heterocyclic ring structures include, but are not limited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, pyrrolidinyl, imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolinyl, or isatinoyl. Also included are fused ring and spiro compounds containing, for example, the above heterocyclic ring structures.
As used herein the term xe2x80x9caromatic heterocyclic ring systemxe2x80x9d refers to a monocyclic and bicyclic heterocyclic ring system containing at least one aromatic ring. The terms xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d as used herein refer to Cl, Br, F or I group. The term xe2x80x9chaloalkylxe2x80x9d and the like, refer to a carbon radical having at least one hydrogen atom replaced by a Cl, Br, F or I atom. A mixture of different halo atoms may be used if more than one hydrogen atom is replaced. For example, a haloalkyl includes chloromethyl (xe2x80x94CH2Cl). Trihaloalkyl includes, for example, trifluoromethyl (-CF3) and the like as preferred radicals.
The term xe2x80x9cmethylenexe2x80x9d refers to xe2x80x94CH2xe2x80x94.
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d as used herein includes salts of compounds of the invention derived from the combination of a compound of the invention and an organic or inorganic acid or base. Preferably, a pharmaceutically acceptable salt of a compound of the invention is a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt, more preferably, a pharmaceutically acceptable base addition salt.
xe2x80x9cPharmaceutically acceptable acid addition saltxe2x80x9d as used herein refers to salts retaining the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid and the like.
xe2x80x9cPharmaceutically acceptable base addition saltsxe2x80x9d as used herein refers to those salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic nontoxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
The term xe2x80x9cbiological propertyxe2x80x9d as used herein means an in vivo effector or antigenic function or activity that is directly or indirectly performed by a compound of the invention that are often shown by in vitro assays. Effector functions include receptor or ligand binding, any enzyme activity or enzyme modulatory activity, any carrier binding activity, any hormonal activity, any activity in promoting or inhibiting adhesion of cells to an extracellular matrix or cell surface molecules, or any structural role. Antigenic functions include possession of an epitope or antigenic site that is capable of reacting with antibodies raised against it.
The term xe2x80x9cisomerxe2x80x9d as used herein refers to a compound having the same number and kind of atoms and hence the same molecular weight as another compound, but differing in respect to the arrangement or configuration of the atoms of the compound. The term xe2x80x9cisomerxe2x80x9d also includes diastereoisomers, enantiomers or mixtures thereof since in the compounds of the invention, carbon atoms bonded to four non-identical substituents are asymmetric. Accordingly, the syntheses described herein may employ racemates, enantiomers or diastereomers as starting materials or intermediates. Diastereomeric products resulting from such syntheses may be separated by chromatographic or crystallization methods, or by other methods known in the art. Likewise, enantiomeric product mixtures may be separated using the same techniques or by other methods known in the art. Each of the asymmetric carbon atoms, when present in the compounds of the invention, may be in one of two configurations (R or S) and both are within the scope of the invention.